There is a need to understand how organ regeneration can be established using stem cell- based therapy. Our goal is to develop clinical approaches for stem cell therapy to regenerate irradiated submandibular glands (SMGs). This is essential to restoring the oral and dental health of head-and-neck cancer patients who suffer from hypofunction after radiotherapy treatment. Our objective is to characterize and expand salivary gland stem cells, and identify conditions to optimize their regenerative capacity in an irradiated environment. The central hypothesis is that a subpopulation of Kit+ cells are stem cells that can be expanded in vitro and then stimulated in vivo after transplantation to regenerate an irradiated SMG. Thus, the following aims will be pursued: 1) characterization of the stem cells in the heterogeneous Kit+ population; 2) in vitro expansion of Kit+ stem cells; and 3) preservation of the stem cell environment in an irradiated gland. This will mainly be achieved by using transgenic mouse strains, cell sorting and transplantation, ChiP-seq and RNA sequencing analysis. The project is innovative because it addresses these aims by using stem cells from both human and mouse SMGs. Altogether, the proposal is expected to significantly progress clinical stem cell-based treatments for long-term SMG regeneration.